Muscle fibrosis is a phenomenon that frequently occurs in diseased or damaged muscle. It is characterized by the excessive growth of fibrous tissue, which usually results from the body's attempt to recover from injury. Fibrosis impairs muscle function and causes weakness. The amount of muscle function loss generally increases with the extent of fibrosis. Fibrosis is usually progressive and can contribute to the patient's inability to carry out ordinary tasks of independent living, such as grasping objects or walking. Fibrosis commonly occurs as a result of muscular dystrophy, as well as due to other afflictions, such as denervation atrophy, a degradation of muscle tissue caused by loss of neural contact to a muscle. For some types of muscular dystrophy, such as Duchenne, fibrosis can result in death as the muscles of the diaphragm are affected (the diaphragm is a skeletal muscle which is involuntary rather than voluntary).
Muscular dystrophies are a heterogeneous group of genetic disorders characterized by the progressive loss of muscle strength and integrity. Dystrophic muscle shows variation in muscle fiber size, infiltration of connective and fatty tissue, and centrally located nuclei. The membranes of the fibers are fragile and extensive damage occurs, leading to necrosis and muscle wasting.
Victims of muscular dystrophies, particularly Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD), frequently suffer from increasing skeletal muscle fibrosis as the disease progresses.
The most common form of muscle dystrophy is the X-linked recessive DMD, a severely penetrating allelic manifestation which affects 1 in 3500 live males at birth; about a third of cases occur as de novo mutations in the infant (Emery A E. (1991) Neuromusc. Disord. 1:19-29).
Usually the disease is diagnosed at 4-5 years of age and by 8-10 years, deterioration of the patient's condition necessitates wheelchair use. By their early teens, further neurological and cardiological symptoms are apparent. Progression of muscle degeneration and worsening clinical symptoms, lead to death in the late teens or early twenties, typically as a result of cardio-pulmonary complications due to fibrosis of the diaphragm.
The leading causes of death in DMD victims, respiratory and heart failure, result from weakness in diaphragm and myocardium muscles that are most affected by fibrosis (Finsterer, (2003) Cardiology 99:1-19). Fibrosis is characterised by an increase in extra-cellular matrix (ECM) constituents especially collagen type I. Both in DMD and Congenital muscular dystrophy (CMD), an increase in type I and III collagens were observed in the skeletal muscle (Hantai et al. (1985) Connect Tissue Res. 13:273-81 and Dunace, et al. (1980) Nature 284:470-472) leading to fibrosis, which correlated with muscle destruction (Zhao, et al. (2003) J. Patho. 201:149-59). The cardiac involvement in DMD is characterized pathologically by degeneration and fibrosis of the myocardium, probably due to myofibroblast activity, centering around the posterolateral wall of the left ventricle.
BMD is a less severe condition than DMD, characterized by slowly progressive muscle weakness of the legs and pelvis, again due to fibrosis of the muscles (although for BMD the skeletal muscles are more greatly affected). The advance of fibrosis often causes ever greater loss of mobility and a reduced life expectancy. At some point, the patient may become too weak to walk and takes to a wheelchair.
Both BMD and DMD are associated with defects in the dystrophin gene, the gene responsible for the production of dystrophin protein, which is a vital part of the dystrophin-glycoprotein complex. DMD is characterized by the near absence of dystrophin protein in skeletal muscles, while BMD results from different mutations in the same gene, resulting in decreased or damaged dystrophin. The presence of some dystrophin protects the muscles of those with BMD from degenerating as badly or as quickly as those of DMD victims.
The dystrophin-glycoprotein complex connects the actin cytoskeleton of myofibres to the extracellular matrix (ECM) and is therefore integral to the contractile structure of muscle (Yue Y, et al. (2003) Circulation, 108:1626-32 and Michele et al. (2003) J. Biol. Chem. 278:15457-60). The preliminary stage of DMD is characterized by the presence of focal groups of necrotic myofibres, muscle hypertrophy and abnormally high levels of muscle creatine kinase (CK). In the pathological phase, repeated cycles of degeneration exhaust the regenerative capacity of muscle-specific progenitor cells (satellite cells) and fibrotic mechanisms cause the progressive replacement of the muscle tissue with collagenous connective tissue (Rafael et al., 1997). These processes lead to joint contraction, loss of ambulation and death from respiratory or cardiac failure (Wells, et al. (2002) Neuromuscle Disord. 12 Suppl 1:S11-22).
The perfect solution for DMD and BMD patients would be to place a normal copy of the dystrophin gene into muscle cells, and hence restore sufficient protein expression to improve structure and function (Khurana, et al. (2003) Nat Rev Drug Discov. 2:379-90). At 3.0 MB the dystrophin gene is vast, and successful therapy would require massive and sustained gene transfer (Hffman, et al. (1987) Cell 51:919-28 and Skuk, et al. (2002) Curr. Opin. Neurol. 15:563-9 and Thioudellet, et al. (2002) Neuromuscul Disord. 12 Suppl 1:S49-51). Muscle fibrosis is a major obstacle in gene therapy since it hampers gene delivery.
An alternative to replacing the faulty gene is to modulate its expression by employing antisense oligonucleotides that alter RNA stability, or splicing (Lu Ql, et al. (2003) Nat. Med. 9(8):1009-14 and Rando T A. (2002) Am. J. Phy. Med. Rehabil. 81(11Suppl):S175-86), thereby resulting in the production of a functional protein. Transplantation of muscle precursor cells (myoblast transfer) has also been explored as a method for restoring dystrophin protein to dystrophic muscle (Law P K et al. (1997) Transplant Proc. 29(4):2234-7). This technique is constrained by the difficulties associated with treating large volumes of muscle with long-lasting effect. An alternative approach is to up-regulate the expression of an endogenous protein that effects some functional replacement (Krag T O, et al. (2001) Acta Physiol Scand. 171:349-58). However, all of these treatments are ineffective unless the progression of the underlying fibrotic condition can be halted or at least ameliorated somewhat.
The crucial role of collagen in fibrosis has prompted attempts to develop agents that inhibit or modulate its accumulation. Several unique post-transcriptional enzymes of the collagen biosynthesis pathway appear to be attractive targets for reducing the formation of collagen fibers or for the accumulation of fibers with altered properties (Prockop D J, (1995) Annu Rev Biochem. 64:403-34).
The major disadvantage of these inhibitors is that they are not collagen-type-specific and may inhibit the biosynthesis of other collagens with serious toxic consequences.
To date there is no effective therapy for reducing skeletal muscle fibrosis. No treatment which affects fibrotic tissue without adversely affecting healthy muscle tissue or other body functions is currently known. The only treatment to have shown clinical efficacy is a prednisone/prednisolone treatment that results in a modest increase in strength, and delays, but does not halt, the progress of the disease (Backman, et al. Neuromuscul Disord. 5:233-41 and Dubowitz, (2002) Neuromuscul Disord. 12:113-6).
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of preventing or retarding the build up of skeletal muscle fibrosis that accompanies disorders such as Duchenne and Becker muscular dystrophies and other muscle dystrophies with extensive fibrosis, as well as to reduce the effect on muscles of the diaphragm for Duchenne muscular dystrophy.
Quinazolinone Derivatives
Quinazolinone derivatives were first taught in U.S. Pat. No. 3,320,124 to American Cyanamid as a treatment for the intestinal parasitic disease, coccidiosis. Halofuginone, (7-bromo-6-chloro-3-[3-(3-hydroxy-2-piperidinyl)-2-oxopropyl]-4(3H)-quinazolinone), an analog of a plant alkaloid originally isolated from the plant Dichroa febrifuga, was described as the preferred quinazolinone derivative. Subsequently, U.S. Pat. Nos. 4,824,847; 4,855,299; 4,861,758 and 5,215,993 all relate to the coccidiocidal properties of halofuginone.
More recently, it was disclosed in U.S. Pat. No. 5,449,678 that these quinazolinone derivatives are unexpectedly useful for the treatment of a fibrotic condition such as scleroderma and graft-versus-host disease (GVHD). This disclosure provided compositions of a specific inhibitor comprising a therapeutically effective amount of a pharmaceutically active compound of the formula:
wherein: n=1-2R1 is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy; andR3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.Pharmaceutically acceptable salts thereof are also included. Of this group of compounds, halofuginone has been found to be particularly effective for the disclosed treatment.
The clinical potential of halofuginone in anti-fibrotic therapy has also been described in Pines, et al. Drug of the Future 21:569-599 and Pines, et al. (1997) Gen. Pharmaco. 30:445-450 and Pines, et al. (2000) Drug Develop. Res. 50, 371-378). Halofuginone, an inhibitor of collagen type I synthesis has been found to inhibit the gene expression of collagen type I, but not of type II (Granot, et al. Biochim Biophys Acta 1156:107-112) or type III (Choi, et al. (1995) Arch Surg 130:257-261).
U.S. Pat. No. 5,891,879 further discloses that the quinazolinone derivatives are effective in treating restenosis. The two earlier-described conditions, scleroderma and graft-versus-host disease, are associated with excessive collagen deposition, which can be inhibited by halofuginone. Restenosis is characterized by smooth muscle cell proliferation and extracellular matrix accumulation within the lumen of affected blood vessels in response to a vascular injury (Choi et al., Arch. Surg., 130:257-261 (1995)). One hallmark of such smooth muscle cell proliferation is a phenotypic alteration, from the normal contractile phenotype to a synthetic one. Type I collagen has been shown to support such a phenotypic alteration, which can be blocked by halofuginone (Choi et al., Arch. Surg., 130: 257-261, (1995); U.S. Pat. No. 5,449,678).
Notably, the in vitro action of halofuginone does not always predict its in vivo effects. For example, as demonstrated in U.S. Pat. No. 5,449,678, halofuginone inhibits the synthesis of collagen type I in bone chrondrocytes in vitro. However, chickens treated with halofuginone were not reported to have an increased rate of bone breakage, indicating that the effect is not seen in vivo. In addition, even though halofuginone inhibits collagen synthesis by fibroblasts in vitro, it promotes wound healing in vivo (WO 01/17531). Thus, the exact behavior of halofuginone in vivo cannot always be accurately predicted from in vitro studies.
Quinazolinone-containing pharmaceutical compositions, including halofuginone, have been disclosed and claimed as effective for treating malignancies (U.S. Pat. No. 6,028,075), for prevention of neovascularization (U.S. Pat. No. 6,090,814), as well as for treating hepatic fibrosis (U.S. Pat. No. 6,562,829), pulmonary fibrosis (WO 98/43642) and renal fibrosis (WO 02/094178), scleroderma and a variety of other serious diseases, exhibit excessive production of connective tissue, which results in the destruction of normal tissue architecture and function.
WO 00/09070 relates to a method for treating and preventing fibrotic process, which results from pathophysiological responses to tissue trauma, preferably cardiac fibrosis.
In most animal models of fibrosis, regardless of the tissue, halofuginone has a minimal effect on collagen content in the non-fibrotic animals, whereas it exhibits a profound inhibitory effect in the fibrotic organs. This suggests a different regulation of the low level house-keeping expression of collagen type I genes on the one hand and the over-expression induced by the fibrogenic stimulus which is usually an aggressive and a rapid process, on the other.
Muscle Tissue
Muscle is a very specialized tissue that has both the ability to contract and the ability to conduct electrical impulses. Muscles are classified both functionally as either voluntary or involuntary, and structurally as either striated or smooth. From this, there emerge three types of muscles: smooth muscle (involuntary), skeletal voluntary muscle (voluntary and involuntary) and cardiac muscle. Skeletal and cardiac muscle are called striated muscle because of their striped appearance under a microscope.
Skeletal muscle may be of the voluntary or involuntary muscle type, being innervated by neurons that originate from the somatic or voluntary branch of the nervous system, providing willful control of the skeletal muscles, or, as in the case of the diaphragm muscles, being controlled by efferent nerves from the respiratory centre which pass down the spinal cord to the diaphragm.
Skeletal muscle cells are long multi-nucleated cylinders, which acquired this characteristic because they develop from the fusion of small single cells into long units. The cells may vary in diameter, averaging between 100 and 150 microns. Skeletal muscle cells are independent cells separated from one another by connective tissue and must each be stimulated by axons of a neuron. All the cells innervated by branches from the same neuron will contract at the same time and are referred to as a motor unit. Motor units vary in size: large motor units with more than 100 cells are typical of the slow acting postural muscles. Very small motor units with around 10 cells or so are typical of fast acting muscles with very precise control such as those which move the eye. Most human muscles have a mixture of motor units of different sizes.
Skeletal muscles have distinct stripes or striations that identify them and are related to the organization of protein myofilaments inside the cell. Skeletal muscle cells are associated with a type of stem cell known as a satellite cell. These cells are believed to aid in recovery of muscle fibers from damage and can contribute their nuclei to replace and supplement the nuclei of the damaged cells. This occurs in response to the “microtears” produced by strenuous exercise and results in increased production of proteins and myofibrils.
Voluntary muscles comprise a variety of fiber types which are specialized for particular tasks. Most voluntary muscles contain a mixture of fiber types although one type may predominate.
Type 1 or slow oxidative fibers have a slow contraction speed and a low myosin ATPase activity. These cells are specialized for steady, continuous activity and are highly resistant to fatigue. Their motor neurons are often active, with a low firing frequency. These cells are thin (high surface to volume ratio) with a good capillary supply for efficient gas exchange. They are rich in mitochondria and myoglobin, which gives them a red color. They are built for aerobic metabolism and prefer to use fat as a source of energy. These are the marathon runner's muscle fibers.
Type 2A or fast oxidative-glycolytic fibers have a fast contraction speed and a high myosin ATPase activity. They are progressively recruited when additional effort is required, but are still very resistant to fatigue. Their motor neurons show bursts of intermittent activity. These cells are thin (high surface to volume ratio) with a good capillary supply for efficient gas exchange. They are rich in mitochondria and myoglobin which gives them a red color. They are built for aerobic metabolism and can use either glucose or fats as a source of energy. These are general purpose muscle fibers which give the edge in athletic performance, but they are more expensive to operate than type 1.
Type 2B or fast glycolytic fibers have a fast contraction speed and a high myosin ATPase activity. They are only recruited for brief maximal efforts and are easily fatigued. Their motor neurons transmit occasional bursts of very high frequency impulses. These are large cells with a poor surface to volume ratio and their limited capillary supply slows the delivery of oxygen and removal of waste products. They have few mitochondria and little myoglobin, resulting in a white color (e.g. chicken breast). They generate ATP by the anaerobic fermentation of glucose to lactic acid. These are sprinter's muscle fibers, no use for sustained performance.
Cardiac muscle is the muscle found in the heart. It is composed of much shorter cells than skeletal muscle that branch to connect to one another. These connections are by means of gap junctions called intercalated disks that allow an electrochemical impulse to pass to all the connected cells. This causes the cells to form a functional network called a syncytium in which the cells work as a unit. Many cardiac muscle cells are myogenic which means that the impulse arises from the muscle, not from the nervous system. This causes the heart muscle and the heart itself to beat with its own natural rhythm. But the autonomic nervous system controls the rate of the heart and allows it to respond to stress and other demands. As such the heart is said to be involuntary.
The cardiac muscle has a number of unique features that reflect its function of pumping blood.                The myofibrils of each cell (and cardiac muscle is made of single cells—each with a single nucleus) are branched.        The branches interlock with those of adjacent fibers by adherens junctions. These strong junctions enable the heart to contract forcefully without ripping the fibers apart.        The action potential that triggers the heartbeat is generated within the heart itself. Motor nerves (of the autonomic nervous system) do run to the heart, but their effect is simply to modulate—increase or decrease—the intrinsic rate and the strength of the heartbeat. Even if the nerves are destroyed (as they are in a transplanted heart), the heart continues to beat.        The action potential that drives contraction of the heart passes from fiber to fiber through gap junctions.        
Due to the numerous structural and functional differences between various muscle types, the effect of an active pharmaceutical ingredient on a particular muscle type cannot be predicted with any degree of reliability according to the effect of that ingredient on a different muscle type.